MARINE ECOLOGY PROGRESS SERIES Vol. 143: 211-223,1996 Published November 14 Mar Ecol Prog Ser

Reproductive ecology of distichus (Phaeophyceae): an intertidal alga with successful external fertilization

Gareth A. Pearson*, Susan H. Brawley

Department of Plant Biology and Pathology, University of Maine, Orono. Maine 04469. USA

ABSTRACT: Patterns of natural release and fertilization success are reported for the open-coast macrophyte Fucus distichus L. (formerly Fucus distichus ssp. distichus), which is restricted to tide pools in the upper intertidal zone. The release and settlement of eggs and of E distichus occurred during daytime low tide periods (DLT), largely on days when the low tide fell between 10:OOand 14:OO h EST; very few were released when high tides fell during the same time interval. Gamete release during nighttime low tides was very low. During the DLT series, gametes were released in all pools and this was better correlated with the time of day than with time of low tide per se. The timing of fertilization showed considerable variation both within and between pools during a sin- gle DLT. No relationship between either pool temperature or osmolality and gamete release was evi- dent, and only small episodes of gamete release coincided with the times of full or new moons. The restriction of gamete release to periods of very low water motion, when tide pools are ~solatedby the low tide, prevents gamete dilution by water exchange and turbulent flows, and results in external fer- tilization success between 78 and loo'%)with low levels of polyspermy (l to 5%). Dispersal of zygotes among tide pools may be facilitated by the low winter temperatures during reproduction, which retard adhesive production and attachment. Our results demonstrate that intertidal organisms, living in habitats characterized by periodic, turbulent flow regimes, Inay achieve high levels of fertilization success by releasing gametes under optimal hydrodynamic conditions for sperm-egg encounters.

KEY WORDS: External fertilization success . Fertilization ecology . Fucoid algae . Fucus distichus . Gamete release . Polyspermy . Rocky intertidal zone

INTRODUCTION umn; fertilization success under these conditions may be less than 1 D/o(Denny & Shibata 1989). Other studies Temporal variation in the supply of propagules (e.g. suggest that even those eggs that are fertilized may zygotes and larvae) to a comn~unitycan play a major still face damage from small-scale shear forces (Mead role in determining its structure (e.g.Gaines & Rough- & Denny 1994). Experimental field studies conducted garden 1985; reviewed by Underwood & Fairweather under less extreme hydrodynamic conditions have 1989). A key component of propagule supply for many confirmed that fertilization success declines with marine is the success of external fertilization, a increasing water velocity (e.g. Pennington 1985, Levi- process that has long been a subject of scrutiny and tan et al. 1992; reviewed by Levitan & Petersen 1995 debate (e.g.Mortensen 1938, Thorson 1950; reviewed and Levitan 1995). However, to our knowledge, there by Levitan 1995). For example, recent modeling stud- have been no previous reports of natural patterns of ies predict that external fertilization is poorly suited to gamete release and external fertil~zationsuccess in an turbulent environments such as the intertidal zone intertidal organism on exposed shores. Fertilization because of rapid dilution of gametes in the water col- success during natural gamete release (spawning) in other habitats has been determined, i.e. in a few marine (Babcock & Mundy 1992, Bab- ' Present address: U.C.T.R.A , Universidade do Algarve, Cam- pus de Gambelas, P-8000 Faro, Portugal. cock et al. 1992, Sewell & Levitan 1992, Babcock et al. E-mail. [email protected] 1994), fish (Petersen 1991, Petersen et al. 1992), and

O Inter-Research 1996 Resale of full art~clenot permitted 212 Mar Ecol Prog Ser

macroalgae (Brawley 1992, Serrao et al. 1996). High MATERIALS AND METHODS levels of fertilization success (ca 75 to 100%) were observed in these studies, except during a proportion Study site and preparation of pools. A total of 8 tide of the rougher days (Petersen 1991, Petersen et al. pools were studied between January and April 1995, 1992), or when ~ndividuals spawned in temporal or 4 In each of 2 moderately exposed sites at Chamber- spatial isolation (Babcock & Mundy 1992, Babcock et lain, Maine, USA (44"N, 69' 30'E). Further studies of 2 al. 1992). Fertilization success was 95 to 100% in the of these pools were conducted during March 1996. Site estuarine, intertidal alga Fucus ceranoides, which A was situated on the eastern side of a rocky promon- mostly releases gametes near slack high tide, and tory (Long Cove Point), and Site B was located ca 50 m which has a semilunar periodicity of release (Brawley further east across the small, south facing bay (Fig 1). 1992). Continuously submerged populations of Fucus Both sites were characterized by granitic rock on sea- vesiculosus in the Baltic Sea also have high (>95%) ward-facing slopes of gentle to medium grade, with fertilization success, and are very responsive to turbu- the pools containing Fucus distichus (formerly F. dis- lent conditions, achieving high fertilization success by tichus ssp. distichus; Rice & Chapman 1985) being not releasing gametes when water velocities are high found just above the Fucus spiralis zone. Observations (Serrao et al. 1996). This suggests that spawning in of immersion time at high tide revealed that the pools marine organisms may be quite sensitive to water at Site A were flushed by waves for approximately 1 h velocity. If so, tide pools on exposed shores offer a tem- longer (ca 4 h) than those at Site B. During neap tides poral refuge from water motion that is particularly pre- coincident with calm seas, pools were sometimes dictable (Denny 1988). observed to be isolated from tidal input for 2 to 3 d. In this paper we describe natural gamete release and Preparation of the sites was carried out between Sep- fertilization success in the fucoid alga Fucus distichus tember and October 1994, prior to the onset of recepta- L., which is a monoecious species found exclusively in cle formation by the adult plants. A tape measure was tide pools in the upper littoral zone of moderately to placed along the long axis of each pool, and at 5 or very exposed rocky shores. Transplant experiments 10 cm intervals (depending on the size of the pool) the with microscopic early stages have demonstrated that distance to either edge was measured perpendicular to this species is physiologically incompetent to survive this axis; water depth was also determined at this time. on emergent rock, except in the lowest regions of the intertidal zone (Chapman & Johnson 1990), which is not its natural distribution. Male and female gametan- gia (antheridia and oogonia) are released from concep- tacles within the reproductive tips (receptacles) of the alga; are spherical, sub-epidermal cham- bers connected via a pore to the surface, and each receptacle contains numerous conceptacles (Evans et al. 1982). Reproduction is seasonal, and the onset of receptacle formation occurs in late autumn in response to short days (Bird & McLachlan 1976). Gamete release occurs during the winter and early spring. Given the variations in temperature and salinity in tide pools dur- ing the winter in New England, USA, and eastern Subtidal I Canada (Edelstein & McLachlan 1975, this study), it is of interest to determine whether gamete release fol- lows the lunar or semilunar penodiclty found in several other macroalgae (Williams 1905, Fletcher 1980, Braw- ley 1992; references in Brawley & Johnson 1992), or occurs in response to different endogenous or environ- mental factors. Further, the synchrony of gamete ----Splash Zone 1 release between Individuals of monoecious species may be lower than that in dioecious species (Brawley 1992) if they are capable of self-fertilization, as mono- ecious fucoids are (Pollock 1970, S. H. Brawley pers. obs.). Our results show that fertilization is successful in Fig. 1. Study area at Chamberlain, Maine, USA (Long Cove F. distichus due to avoidance of gamete release under Point), showing the positions of tide pools containing Fucus turbulent conditions at high tide. distichusat Sites A and B Pearson & Bra~vley:Keproduct Ive ecology of Fucus distichus 213

film canister carefully onto the lid to seal the sample before lifting the assembly from the pool. The simplic- ity and rapidity of this system were advantages when working at low temperature, as samples were often collected from under thick ice cover. Sampling and field conditions. Daily sampling of eggs and zygotes was performed prior to immersion of the pools by a daytime or evening high tide (i.e. fol- lowing a daytime low tide). The disk assemblies were replaced with fresh ones on each occasion. Unlike many invertebrates, there is no larval stage in the life history of fucoid algae, and therefore settlement of eggs and zygotes into the shallow water of these iso- lated tide pools during the period of low tide directly reflects gamete release. Breaks in the daily sampling regime in 1995 occurred between January 31 and Feb- ruary 10 and between February 17 and 21. In addition, ice cover prevented sampling on several occasions when either (1) algae overlying settlement disks were embedded in the ice, preventing their removal to reach samples beneath, or (2) settlement disks were them- selves embedded in ice. On returning to the laboratory, Fig. 2. Settlement disk positioned in the substrate. The disk is held in place in a canister Lid that fits flush to a collar of Poxy both the settlement disk and the seawater in the canis- Putty, both flush with the substrate. The weighted bolt pro- ter were examined with a dissecting microscope, and trudes into the central hole to anchor the assembly. The the number of eggs and zygotes was recorded (during assembly is removed by snapping the canister onto the 11d to peaks of settlement, subsamples were counted to pro- retain the sample, and lifting it from the pool vide estimates of settlement per disk). At the same time that disks were retrieved, air and pool water tempera- Maps of each pool were printed on paper, cut out, and tures were recorded, and samples of water from the weighed to allow the surface area to be estimated from top and bottom of each pool were removed with a 1 m1 the weight of paper of known area. Percentage cover syringe and transferred to microcentrifuge tubes for of F. distichusover the entire area of each pool was measurement of osmolality. Osmolality of water sam- estimated using a 50 x 50 cm quadrat subdivided into ples was determined in the laboratory using a Wescor 10 X 10 cm squares. 5500 vapor pressure osmometer (Logan, UT). Irradi- During low tide the pools were drained, and sites for ance measurements in the field or at the Darling settlement disks were selected haphazardly within Marine Center, ME (6 miles from field sites) were patches of plants (n = 3 per pool). Settlement disk recorded between 12:OO and 14:30 h EST (Eastern assemblies consisted of a film canister lid (diameter Standard Time; hereafter all times given in EST) using ca 38 mm) into which Sea Goin' Poxy PuttyT\' a Li-Cor L1 188B quantum meter with spherical sensor. (Pern~alitePlastics, Newport Beach, CA) disks (diame- To resolve settlement patterns on a finer temporal ter 28 mm) were fitted; these were held together with scale, settlement disks (on lids) were placed haphaz- a bolt (Fig. 2). Holes were drilled into the rock with a ardly among algae in Pools A2, A4, B3, and B4 during gas-powered drill (Ryobi Ltd, Hiroshima, Japan): a single low tides on 2 occasions (January 26, low tide = shallow hole at the surface that was large enough for 12:29 h; and January 30, low tide = 16:25 h). Following the settlement disk (4 to 5 cm diameter), and a smaller, isolation of the pools by the receding high tide, sam- deeper hole in its center (ca 1 cm diameter) into which ples were collected at 90 min intervals until the follow- the weighted bolt protruded (Fig. 2). The shallow hole ing high tide. Water motion was essentially absent dur- was filled with Poxy PuttyTM,with care taken to avoid ing the sampling period, so disks were not anchored to filling the deeper hole. A film canister lid was covered the substrate, but sample collection was otherwise as with a thin film of silicone grease and pushed into the described above. Poxy PuttyTMuntil flush with the surrounding rock sur- Gamete release and settlement during daytime low face.After the Poxy PuttyT)Vried,the lid was removed tides (DLT) and nighttime low tides (NLT) were deter- to leave a depression into which disk assemblies could mined in March 1996 in 2 pools (A2 and B4) by placing be fitted. This design allowed sample retrieval under- settlement disks haphazardly among algae with water with a minimum of disturbance, by snapping a mature receptacles, as described above. These data Mar Ecol Prog Ser 143: 211-223, 1996

were log-transformed and analysed by repeated- removal was designated t = 30), by which time a suffi- measures ANOVA (Systat Inc. version 5.2.1) and clent quantity of gametes for analysis had been means were compared using Tukey's test. released. Subsamples of eggs and zygotes were fixed Release of eggs in Pool B4 was compared at low and in acetic acid:ethanol (1:3) at regular time intervals, high tide by placing fertile branches (n = 6 to 8 recep- stained and examined as described above (n = 25 tacles) in 50 m1 polypropylene centrifuge tubes with eggs). These data were used to construct calibration the side walls replaced by 40 pm nylon mesh to retain curves, from which the timing of fertilization in the fucoid eggs (ca 70 pm diameter). During neap tides on field was estimated as described by Brawley (1992). March 25 and 26 pools did not receive tidal input, Stages of karyogamy were assigned a stage (A to E) whereas on April 1 and 2 the pools were flushed at after Brawley (1992). high tide. Tubes containing fertile branches were teth- Laboratory culture of Fucus distichuszygotes. ered to bolts in pools for the sampling period, after Receptacles of Fucus distichus released gametes into which the receptacles were removed for fresh weight filtered seawater after 3 to 4 h of illumination. The determination, and the tubes resealed and transported zygotes were passed through 125 pm nitex to remove in seawater back to the laboratory to count eggs. oogonia, washed 3 times in filtered seawater, allowed Fertilization success, time of fertilization and poly- to settle on replicate glass cover slips in 60 mm Petri spermy analysis. Levels of natural fertilization success dishes, and cultured at 0 or 5°C with 80 pm01 photons and polyspermy (i.e. the fertilization of an egg by more m-* S-' irrad~anceand a 12 h light:12 h dark photo- than one sperm; a lethal condition) in the field were period. Development and adhesion of zygotes and essentially determined as described by Brawley (1992). embryos on replicate cover slips were monitored at Sea Goin' Poxy PuttyTMplates (28.3 cm2) were placed regular intervals with a stereozoom microscope. in upturned lids of polypropylene containers of an Analyses are based on counts of 30 to 40 zygotes per appropriate size. These were placed haphazardly replicate coverslip. among algae in the pools during periods of gamete release, following which the container was carefully placed over the lid. After being removed from the pool, RESULTS the sample was quickly filtered through 40 pm nylon mesh to retain eggs, and the filter plus a minimal vol- Patterns of gamete release and settlement in the field ume of seawater (to prevent bursting of unfertilized eggs) was transferred to acetic acid:ethanol (1:3)for Antheridia containing sperm and oogonia containing fixation in the field. This procedure took less than eggs were shed onto the surface of receptacles and 5 min per replicate. A different method was used on then settled directly below the point of release, March 10, 11 and 26: Samples were aspirated directly because there was little (if any) water motion in the from the pools with a Pasteur pipette and placed in fix- pools; oogonia, eggs, zygotes and antheridia are nega- ative in small vials. In the laboratory, fixed eggs and tively buoyant, and sperm are negatively phototactic. zygotes were filtered through 125 pm nylon mesh to During spring tides (i.e. high-amplitude tides occur- remove large detritus, stained with Wittman's aceto- ring in association with full and new moons), the pools iron hematoxylin (Wittman 1965) and examined under at both sites were flushed by wave action around high the microscope to determine the presence or absence tide, and during such times populations of Fucus dis- of sperm pronuclei (Brawley 1987; N.B. eggs contain- tichuswere subjected to highly turbulent flow regimes. ing more than one sperm pronucleus are polyspermic). Pools at Site A were generally flushed ca 30 min earlier When the sperm pronucleus was in the same plane of on the rising tide than those at Site B. However, during focus as the egg nucleus, the distance of both from the neap tides, pools at both sites were isolated for periods egg surface was recorded using a calibrated ocular up to 3 d. Settlement varied between pools over ca 2 micrometer. From these data the time of fertilization, orders of magnitude (Fig. 3; cf. Pools A2, B1 and B4). relative to the time of fixation, was determined from a The areas of pools were between 0.36 (B3) and 4.81 m2 calibration curve in the laboratory (see below. 75 (B4) and percentage cover varied from 8.7 (A4) to zygotes were counted per sample; n = 3 or 5 samples 43.4 % (A2),but additional data are required to assess per pool). the effects of pool size or cover on the magnitude or The rate of sperm pronuclear migration towards the timing of gamete release. The position of the settle- egg pronucleus and the stage of karyogamy were ment disks relative to the largest reproductive adults in determined in the laboratory at both 3°C and 8°C. a patch was an important source of variation since eggs Receptacles in seawater were incubated in the light in and zygotes settling during low tide fell vertically a walk-in culture chamber and were removed within below the point of release. The distribution of F. dis- 30 min of the onset of gamete release (the time of tichus within each pool was highly contagious, as Pearson & Brawley: Reproductive ecology of Fucus distichus

4000 - Pool A4 3000 -'.<.-.r.,r. ,C . ': -e 2000 -

mma6%:$s3z$$3zzzCCC sM~gr,C=z~r,COh-7777LL". N N -? Date Date

Fig. 3. Settlement of eggs and zygotes (-) of Fucus djstjchusbetween January 15 and March 16, 1995 (n = 3 settlement disks, except Pool Al, where n = 2; mean i SE),and the temperature of water in the pools at the time of sampling (--a--) for (a) Site A and (b) Site B. Lunar phase is shown along the top; blocks on the x-axis indicate days on which the low tide fell between 10:OO and 14:OOh, and on which most settlement events were recorded. For the settlement data, the thin dotted line represents delib- erate gaps in sampling; other missing data resulted because of excessive ice cover on pools shown by values of the variance to mean ratio that full and new moon (e.g.January 30 in Pools A2, B1 and exceeded 20 for each pool [data not presented; a value B4; February 14 in Pool A2; February 16 in Pools B2 of 1 indicates a random distribution, and increasingly and B3; February 17 in Pool Bl). higher values indicate more contagious distributions The major period of gamete release observed during (Pielou 1977)l. 1995 occurred in March (Fig. 3). Gametes continued to Settlement was generally synchronous in popula- be released into early April (data not shown), followed tions both within and across several pools, suggesting by rapid necrosis and loss of receptacles during mid to that common proximal factors influenced release of late April, prior to a resumption of vegetative growth. It gametes at these sites (Fig. 3). For example, on Janu- is likely that the increasing level of gamete release ary 26 (A2, A4 and B4) and 30 (A2,B1 and B4),on Feb- from January to March was a result of receptacle mat- ruary 11 (Al, A2, A4 and B4), and particularly on uration, although gametangia appeared to be mature March 13 (A1 to 4 and B2 to 4), pools shared synchro- throughout this period. nous gamete release and settlement events. DLT is a There was a large temporal and spatial variation good indicator of gamete release (as determined from in the osmolality of the pools relative to seawater (ca settlement), since 28 of the 37 settlement events 1000 mM kg-') during the sampling period, due to both recorded for all of the pools during 1995 occurred climatic conditions and tidal periodicity (Fig. 4). Verti- when the DLT fell between 10:OO and 14:OO h, and a cal salinity gradients were formed in 2 ways: as water further 4 occurred within 2 d of this 'reproductive win- froze at the surface, salts were concentrated in bottom dow'. Major peaks in gamete release and zygote set- water relative to the surface; secondly, input of fresh tlement were not correlated with lunar phase, water by precipitation (rain or fog) caused the salinity although minor episodes occurred around the time of of the water near the surface to decrease, but had only 216 Mar Ecol Prog Ser 143: 211-223, 1996

,600ip001 A1 1600; -p001 B1 : Wypa, - - - '- - . 0 - -' ---. - 0 i- -- - - Pool B2 Pool A2 c -- - 1600-

: Pool B3 3200; I: z 0- -- I U- . . m - A3 Pool 1 2400. . ..

1600:

0 - 0

-- 2400; Pool B4 I ---L

Date Date Fig. 4. Osmolality of water in tide pools at (a) Site A and (b) Site B, between January 15 and March 16, 1995. Samples of surface (a) and bottom water (0) were taken near the end of a low tide period. The horizontal broken line represents the osmolality of normal seawater (ca 1000 mM kg-'); vertical broken lines show dates on which gamete release was recorded for the particular pool. Blocks on the x-axis show days on which the low tide fell between 10:OO and 14:OO h

a minor effect on salinity near the bottom. The maxi- Pool temperature during isolation of the pools at low mum recorded osmolality at the bottom of the pools tide varied between -5°C (under thick ice cover on was 3540 mM kg-' (Pool B3, March 13) and exceeded February 14) and +8"C (March 8), while the mean sea 1500 mM kg-' in all of the pools periodically. The min- surface temperature during the sampling period was imum recorded osmolality of water at the pool surface 5.1°C in January, reaching a low of 3.2"C in February was 63.5 mM kg-' (Pool Al, March g), and frequently before rising slightly to 3.4OC in March. High levels of dropped below 500 mM kg-' in all of the pools (Fig. 4). gamete release were recorded at temperatures be- During neap tide periods that coincided with DLT, tween -l and +?"C,and 29 of the 38 events occurred at pools often did not receive tidal input for several days, temperatures above freezing. The temperature of the suggesting that osmotic stimuli might also influence pools may, therefore, be correlated with settlement, gamete release. For example, none of the pools was although at temperatures significantly below zero the flushed during high tide between March 10 and 12 and formation of ice causes: (1) increased salinity and a modest amount of settlement was recorded in most of (2) trapping of receptacles (precluding gamete re- the pools. Then, on March 13, the morning high tide lease); without experimental manipulations, the effects flushed all the pools in Site A as well as Pool B4 (the of these factors cannot be resolved from the effects of latter receiving only limited wash by larger waves), temperature per se. Temperature has a marked effect and in each case, high settlement was recorded. It on the adhesion of zygotes to the substrate; following should be noted, however, that significant numbers of culture for 30 h at 5°C in the laboratory 69.3 ? 2.9% eggs and zygotes were released in pools B2 and B3 on (SE) of zygotes had attached, whereas this figure was these days in mid March, and these pools were not reduced to 20.9 + 2.7 % at O°C (n = 14 coverslips). washed by waves on March 13. Other than the possible No relationship between the level of irradiance and coincidence (in some pools) of gamete release with patterns of gamete release was evident. Although large osmotic changes on several days in mid March, some release events occurred on sunny days, when however, gamete release was not related to osmotic irradiance was typically ca 2500 pm01 photons m-2 S-' changes. (e.g. January 26 and 30), others occurred at intermedi- Pearson & Brawley: Reproductive ecology of Fucus distichus 217 - =-

Jan 26. 1995

Jan 30, 1995

Time interval (EST)

Fig. 5. Settlement of eggs and zygotes of Fucus distichus in Pools A2 and 04 on January 26 (a and c), and A4 and B3 on January 30, 1995 (b and d) during single DLTs (n = 5 settlement disks, mean  SE). Samples were collected every 1.5 h after pools were isolated by the receding high tide ate (March 8, 997 pmol photons m" s") or low irradi- B4 (FIl0= 39.67; p < 0.001). Comparisons of means ance (March 13, 173 pmol photons m" s"'). showed that within a pool release during NLT on Temporal patterns of egg and zygote settlement dur- March 11/12 and 12/13 was lower than DLT on March ing a single DLT on January 26 and 30 are shown in 12 and 13 except in Pool B4, where release during NLT Fig. 5. On January 26, sampling began at 08:OO h, after of March 12/13 was not significantly lower than the pools were isolated on the falling tide following the release during DLT on March 13 (following the peak of 06:OO h high tide. Settlement was nearly absent until release on March 12). the 11:OO to 12:30 h sampling period, but high levels of settlement occurred after the 12:30h low tide, between 12:30 and 15:30 h (Fig.5a, b).This pattern was found in each of the 4 pools studied. On January 30, settlement occurred over the same time period as on January 26; settlement began during the first sampling period after the 10:OO h high tide, between 12:OO and 13:30 h, and reached a peak between 13:30 and 16:30 h (Fig. 5c, d). On both days the range of pool temperatures recorded was similar (-3 to + 1° on January 26 and -2 to O° on January 30), and although no irradiance data for these 2 d are available, the weather was sunny and clear. These data suggest that settlement (and therefore gamete release) coincides with DLT, not with a partic- ular phase of the tidal cycle. Date During peaks of release associated with DLT on March 12 (09:49 h) and 13 (10:55 h) 1996, gamete Fig. 6. Egg release by Fucus distichus during DLT and NLT in Pools A2 and B4 between March 11 and 16, 1996. Sampling release during NLT was very low (Fig. 6). Repeated- intervals were the periods between immersion by the high measures ANOVA indicated significant differences be- tide. Alternating light and dark blocks represent day and tween release during DLT and NLT for Pools A2 and night. Values are means  SE (n = 6 settlement disks) Mar Ecol Prog Ser 143: 211-223, 1996

orders of magnitude lower than during DLT on March 26, and average egg release was less than 20 eggs g-' fresh wt during calm periods following the high tide on both days (Fig. 7b). These data: (1) support the hypoth- esis that gamete release occurs primarily during DLT periods; (2) show that gamete release is very low when the pools are being flushed by the high tide during this interval; and (3) are in agreement with data from labo- ratory experiments showing a strong inhibition of I Mar 25 Mar 26 gamete release by water motion in Fucus distichus (SerrBo et al. 1996).

Fertilization success and polyspermy

Fertilization success and levels of polyspermy in replicate samples of 50 to 100 eggs and zygotes were determined on 4 dates: January 30, March 10, l1 and 26 (Table 1).On each date, samples were collected 3 to 4 h after low tide, several hours after receptacles were observed to be releasing oogonia and antheridia. An Apr 1 Apr2 additional sample was collected on March 26, between Date 12:00 and 12:40 h (prior to low tide), to investigate the changes in fertilization level over the settlement Fig. 7. Egg release by Fucus distichusduring (a) DLT (March 25 and 26, 1995) and (b) DHT (April 1 and 2, 1995). Hatched period. Because gamete release was high in most of bars are samples taken during isolation of the pools from the pools on March 26, eggs and zygotes could be wave actlon (low tide), and shaded bars are samples taken pipetted directly from the area of Poxy PuttyTMsur- when pools are subjected to wave action (high tide). Values rounding the settlement disks, obviating the need to k are means SE (n = 6 nitex bags) place fertilization plates in the pools on this date. How- ever, since low settlement was observed in certain Settlement might underestimate gamete release dur- pools, only 1 replicate was obtained from Pools B1 and ing turbulent periods of wave exposure at high tide, B2 at both sampling times, and from Pool B3 at 12:20 h because of the potential for sample loss due to water (Table 1). Fertilization success in samples taken 3 to motion. Therefore, gamete (egg) release was mea- 4 h after low tide was 96.8 to loo%, except on March sured directly during times when low tide versus high 10 and 11 when fertilization success in Pool B4 was tide occurred in the middle of the day in order to com- between 78 and 79%. The reason for these slightly pare gamete release in calm and turbulent conditions lower values is not clear; however, the pool tempera- during this period. Egg release was measured in Pool ture on March 10 and 11 was lower than that on March B4 on days coinciding with DLT (March 25 and 26) and 26 (3, 6 and 10°C respectively), which may have daytime high tides (DHT; April 1 and 2), as shown in affected the rate of gamete release from oogonia and Fig. 7. On March 26, egg release was high between antheridia (Quatrano 1980). Thus, it is possible that 10:OO (when release of gametangia onto the surface of eggs were continuing to be fertilized at the time the receptacles was quite apparent) and 13:OO h, prior to samples were taken on March 10 and 11. On January the low tide at 13:OO h. Between 13:00 and 16:OO h, egg 30, the maximum temperatures of Pools A2 and B4 release was more than 2 orders of magnitude lower were low (2.5 and 3.0°C, respectively), but samples (Fig. ?a). On March 25, egg release was only deter- were taken later in the evening, since the high tide mined between 13:00 and 17:00 h and therefore it is was later. On March 26, fertilization levels of 66.3 to uncertain if higher release occurred earlier in the day, 100% were found in samples taken prior to lowest low although little gametangial release from individuals in tide, and these values increased in the same pools to the pool was observed visually, suggesting that this between 99.6 and 100% when samples were collected was not the case. March 25 and 26 fell during a neap 4 h later (Table 1). tide series, and the pools at Site B were not washed by The estimated timing of fertilization in the field the high tide on either day; therefore, there were no revealed differences between the samples from Janu- periods of turbulence during the tidal cycle. Egg ary 30 and March 26. On January 30, fertilization in release during DHT on April 1 and 2 was more than 3 Pools A2 and B4 occurred in the late afternoon or early Pearson & Brawley: Reproductive ecology of Fuc~lsdistlchus 219

Table 1. Fert~lizationsuccess and polyspermy levels in samples of eggs and zygotes of Fucus djstichus collected from tide pools at Chamberlain, Malne, between January and March1995. For each sample (pool),n replicates of between 50 and 100 eggs per zygote were counted. The site (A or B), pool and date are shown, together with the time of collection (EST) in relation to the day- time low tide (LT). The estimated range of times of fertilization in field samples was obta~nedfrom laboratory callbrations of sperm pronuclear migration rate and stage of karyogamy nd: not determined

Pool Date LT Fixation n 'X'Fertilized Estimated t~me '% Polyspermic time mean (SE) of fertilization mean (SE)

Jan 30 16:30-17:30 Jan 30 18:OO h Mar 10 nd Mar l1 nd Mar 26 08:30-11:30 h Mar 26 Before 12:30 h mar26 08:30-10:OO h Mar 26 Before 12:30 h Mar 26 After 11:15 h Mar 26 Before 12:OO h Mar 26 11:oo h Mar 26 Before 12:OO h Mar 26 11:30 h Mar 26 12:30-13330 h Mar 26 ll:30-12:OO h Mar 26 11:30-13:30 h evening 1 to 2 h prior to sample collection, whereas the timing of: (1) gamete release from individual adults, majority of eggs were fertilized before noon on March (2) breakdown of oogonia and antheridia, and/or 26, and fertilization was complete before 12:30 h in 4 of (3) fertilization processes such as sperm motility and the 6 pools sampled (A2, A3, B1 and B2), and by sperm dispersal (availability); it is not an artifact due 13:30 h in the remaining 2 (B3 and B4). The rate of to variation in sperm pronuclear migration rate. The sperm pronuclear migration and stage of karyogamy timing of fertilization was reconstructed in samples of were temperature-insensitive in laboratory calibra- eggs and zygotes fixed at a discrete point in time dur- tions at 3°C (Fig. 8) and 8°C (data not shown), repre- ing the low tide (18:30 and 19:OO h on January 30; senting temperatures encountered in the field when 12:30 h on March 26). The time at which fertilization collecting samples for analysis of fertilization success. occurred in each zygote sample was calculated from Therefore, any variation in the timing of fertilization the calibration curve (Fig. 8) of sperm pronuclear between san~ples in the field reflects variation in migration. On both days, fertilization in Pool A2 (Fig. 9a, c) began earlier and occurred over a broader time interval than in Pool B4 (Fig. 9b, d).The data from Pool A2 show considerable variability both within and between replicate sampling sites, e.g. 60, 9 and 5% of zygotes collected on March 26 at the 3 sampling sites had been fertilized for >4 h prior to fixation. This inter- esting variability between replicates could result from: (1) a difference in the time of fertilization of eggs which are available to be fertilized (e.g. due to variation in sperm concentration), or (2) variation in the time of release of gametes by individuals across Pool A2. Sampling of gametes (eggs and sperm) and zygotes at Time After Gamete Release (min) intervals throughout the period of gamete release and Fig. 8. Laboratory calibration of sperm pronuclear migration fertilization is required to distinguish between these 2 and stage of karyogamy with time at 3°C in Fucus dist~chus. possibilities. Receptacles were placed into seawater at time = 0 min, and Polyspermy was low in all samples, typically 1 to 3 % removed following gamete (eggs and sperm) release 30 min except in Pool B4 on March 11, where it was 4.9 %. Set- later. Aliquots were flxed at 30 to 60 min intervals for 7 h. Zygotes were stained with aceto-iron haematoxylin to deter- tlement on January 30, and on March 10 and 11 was mine the positions of the sperm and egg pronuclei and stage not high (Fig. 3), and it could be argued that poly- of karyogamy. Values are means i SE (n = 25) spermy was low on these occasions because the sperm 220 Mar Ecol Prog Ser 143: 211-223, 1996

Jan 30, 1995

T~me(EST) T~me(EST)

Mar 26, 1995 - 60 E j d Fig. 9. T~meduring emer- - 50 m . - sion of tide pools A2 (a 40 ++.. . ; 0 and c) and B4 (b and d) -C ... .. K when eggs of Fucus djs- d 30 a, 1 30 g tichus become fertilized N 'I 2 Data are for January 30 $ 20 . .. . . ; .20 $ (a and b) and March 26, - ..::...... :.. ' : g, 10 .. .: .. 1995 (c and d). Values are m W means + SE of n = 5 (Jan- o uary 30) or n = 3 (March S g6 ss &E 88 gg g: g$ 83 $S 89 26) samples (75 eggs per $ *$ g" +"B= er NN =C ,, sample) T~me(EST) T~me(EST) concentration in the pools was relatively low. How- tllization occurred more rapldly during the low tlde ever, on March 26 a visual assessment suggested that penod in March samples than samples taken in Janu- both gamete release and settlement on disks was very ary These obse~vationssuggest either that receptacle high (Fig. 7), while polyspermy was low (Table 1). matunty Increases through the reproductive season, or Therefore, the available data suggest a low incidence that env~ronmentalfactors such as pool temperature of polyspermy in Fucus dlstichus, even when gametes are more favorable for gamete release and fe~t~llzat~on are released into relatively low volumes of calm water at th~stime in the pools. The good agreement found between penods of gamete release and days on whlch low tide fell be- tween 10 00 and 14 00 h suggests that an absence of DISCUSSION water motion and a period of lrrad~anceare both nec- essary for release Thls IS further supported by the lack The data presented in this paper show that the of gamete release dunng NLT Both endogenous fucoid seaweed Fucus distichus L. is able to achieve rhythms (e g circadlan ones) and proxlmal environ- high levels of fertillzatlon success In the intertidal zone mental factors could contribute to the timlng of gamete (78 to 100%) by releasing gametes synchronously dur- release Almost no shedding occurred on days coincld- ing periods of very low water motion in iide pools so- lng wlth daytlme high tldes, particularly dunng the lated at low tide. Major penods of gamete release were period when the pools were be~ngwashed by waves not associated with the lunar or tidal phase, in contrast Endogenous rhythms of algal gamete release have to several other seaweeds (Williams 1905, Hoyt 1927, been demonstrated prev~ouslyIn a few specles (Muller Christle & Evans 1962, Fletcher 1980, Norton 1981, 1962, Philllps 1988) although In other specles gametes Brawley 1992); instead, they occurred when the DLT are released only in response to envlronmental cues was between 10:00 and 14:00 h. Following release and associated with tldal perlodiclty (Ohno 1972 further fertilization during low t~de,zygotes remain un- references in Brawley & Johnson 1992) We have attached and potentially subject to dispersal for a recently shown that receptacles of Fucus dlst~chuscol- period of at least 1 tldal cycle at the low temperatures lected 2 d pnor to the onset of DLTs, and placed in cul- recorded durlng the reproductive season. The magni- ture under constantly calm or simulated tidal condi- tude of gamete release was greatest in March, and fer- tlons, released gametes only with the onset of DLTs Pearson & Brawley: Reproducti\le ecology of Fucusdistichus

and gamete release in the field (Serrao et al. 1996). nington 1985, Denny & Shibata 1989, Levitan et al. However, gamete release was suppressed during this 1992). A small amount of water motion should be ben- period if cultures were subjected to high water motion, eficial for fertilization, because it ensures adequate and photosynthetic competence under calm conditions mixing of gametes (Denny & Shibata 1989). However, was required for release (Serrao et al. 1996). it may be less important for F. distichus, which is a Other than water motion and light, none of the envi- monoecious seaweed capable of self-fertilization in ronmental variables measured in this study is a good which sperm are actively attracted to eggs by a predictor of gamete release. However, several of the pheromone (Maier & Miiller 1986). Following the larger release events in March occurred coincidently release of gametangia from the conceptacles, there is a with above average daily pool temperature (measured short period during which gametes remain associated near the end of low tide). Higher temperatures would with the parent receptacles, since a quantity of muci- increase the rate of gamete maturation and any lage is released together with the gametangia. Pre- physiological processes involved in gamete release, sumably, during this period eggs are closely associated although gametes were also released at temperatures with sperm from the same parent, as well as sperm as low as -l°C, well below the ambient sea surface from receptacles of neighbors when densities of indi- temperature. In addition, gamete release may be indi- viduals are high. Eggs are fertilized over a period of rectly affected by low temperatures, which cause ice to >4 h in some pools, providing some support for our cover the pools during their isolation at low tide, and prediction that gamete release in monoecious, self- therefore reduce irradiance and increase salinity. fertilizing species might be less synchronous between Gamete release was generally low when large salin- individuals than for dioecious species. Fucoid algae ity gradients were present in the pools (but see data for should prove to be excellent model organisms in which Pool B4 on March 11; Figs. 3b & 4b). Similar variations to test such hypotheses in future studies. The high lev- in salinity are experienced by Fucus distichus popula- els of fertilization success found in this study (78 to tions in Nova Scotia, Canada (Edelstein & McLachlan 100%) are similar to those found in dioecious species 1975), although the maximum values found by these of fucoid algae from estuaries (Brawley 1992) and the authors were exceeded in this study. Both hyper- and Baltic Sea (Serrao et al. 1996). hypoosmotic conditions can potentially affect gamete The results of this study raise 2 intriguing questions release and fertilization, since receptacles from a sin- concerning gene flow in Fucus distichus. The first con- gle alga may be near both the top and bottom of a cerns the extent to which self-fertilization occurs, and pool's water colun~n.After settlement, potential hyper- the second concerns the ability of zygotes to disperse osmotic stress is more important, because zygotes between tide pools. Because eggs and sperm are remain near the bottom of the pools: it is known that released simultaneously by F. distichus into a re- osmolalities within the range of those measured in the stricted volume of very calm water, sperm limitation is tide pools inhibit the expression of polarity in Fucus unlikely to occur. However, sperm competition is likely vesiculosus for up to 14 d (Torrey & Galun 1970). In to strongly favor self-fertilization because eggs would their experiments, Torrey & Galun used sucrose as be closer to their sibling sperm than to more distant osmoticum, but zygotes in seawater of similar osmo- potential competitors (Grosberg 1991, Petersen 1991, lality will presumably be exposed to the added stress of Yund & McCartney 1994, Levitan & Petersen 1995, potentially cytotoxic intracellular concentrations of Yund 1995). Further studies to look at gene flow within ions. Acclimation of adult F. distichus to hyperosmotic pool populations, with the aid of molecular genetic conditions has been shown to contribute to increased techniques (allozyme or nucleic acid markers), will be freezing tolerance in laboratory studies (Pearson & necessary to determine the relative proportion of self- Davison 1994).Zygotes can be maintained at 0 and 5°C fertilization versus out-crossing. Whether or not self- with limited development for weeks, or even months fertilization limits gene flow between individuals, the (McLachlan 1974), but the physiological and develop- restriction of gamete release to low tide periods means mental consequences of combined low temperature that individuals within a form an isolated and high salinity have not been investigated for early breeding population, and gene flow between pools is developmental stages. largely dependent on the dispersal of zygotes. The low The ability of Fucus distichus to synchronize gamete temperatures encountered by reproductive F. distichus release during periods of low (near zero) water motion result in slow rates of adhesive production by zygotes, is an extremely valuable adaptation for an organism potentially allowing 1 to several high tide periods dur- inhabiting the intertidal zone. Both modeling ing which dispersal to neighboring tide pools could approaches and experimental field studies of induced occur. The success of dispersal and the scale over spawning have shown that fertilization success which it may effectively occur are not known, how- declines rapidly with increasing water motion (Pen- ever, and must await future investigation. 222 Mar Ecol Prog Ser 143: 21 1-223, 1996

The first event in the life history of Fucus distichusis Denny MW (1988) Biology and the mechanics of the wave the production of zygotes by external fertilization. Suc- swept environment. Princeton University Press, Princeton cessful external fert~lizationis achieved by synchroniz- Denny MW, Shlbata MF (1989) Consequences of surf-zone turbulence for settlement and external fertihzation Am ing gamete release with periods of very low or zero Nat 134:85cl-889 water motion in tide pools isolated by the low tide, and Edelstein T, McLachlan J (1975) Autecology of Fucus djs- does not occur during periods of high water motion at tich~lsssp.distichus (Phaeophyceae: ) in Nova Sco- high tide. These data also suggest that F. distichushas tia, Canada. Mar Biol 30:305-324 Evans LV, Callow JA, Callow bIE (1982) The biology and b~o- an endogenous rhythm that permits gamete release to chemistry of reproduction and early development In occur under the most favorable conditions; calm condi- Fucus. In: Round FE, Chapman DJ (eds) Progress in phy- tions alone do not stimulate gamete release in this spe- cological research. Vol 1. Elsevier Biomedical Press. Ams- cies (Serrdo et al. 1996; these results). Moreover. calm terdam, p 67-110 conditions during DLTs are required. Finally, these Fletcher RL (1980) Studies on the recently introduced brown alga Sargassum muticum (Yendo) Fensholt. 111. Periodicity observations suggest that models predicting low exter- in gamete release and 'incubation' of early germling nal fertilization success in intertidal organisms do not stages. Bot Mar 23:425-432 apply to tide pools and possibly other situations (e.g. Games S, Roughgarden J (1985) Larval settlement rate: a slack high tide) where water motion in the envlron- leading determinant of structure in an ecological cornmu- nity of the marine intertidal zone. Proc Natl Acad Sci USA ment varies predictably in time. 82:3707-3711 Grosberg RK (1991) Sperm-mediated gene flow and the genetic structure of a population of the colonial ascidian Acknowledgements. We thank Tim Mlller and the staff at the Bolryllus schlosser~.Evolution 45:130-142 Darling Marine Center, who facilitated much of thls work. Hoyt WD (1927) The penodic fruiting of Dictyota and its rela- Thanks for help from Cathy and Alec Pearson in preparing tion to the environment. Am J Bot 14.592-618 the study site, and to Ester Serrao for help with collection of Levitan DR (1995) The ecology of fertilization in free-spawn- settlement and field fertilization samples. Thanks to Dr Susan ing invertebrates. In: McEdward L (ed) Ecology of marine Williams and 2 anonymous reviewers for suggesting several larvae. CRC Press, Boca Raton improvements to an earlier version of the manuscript. 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This article was submitted to the edit01 Manuscript first received: February 14, 1996 Revised version accepted: August 9, 1996